COMBINATION, THERAPEUTIC USES AND PROPHYLACTIC USES

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a U.S. National Phase of International Patent Application No. PCT/NZ2023/050010, filed Feb. 13, 2023, which claims the benefit of New Zealand Patent Application No. 785100, filed Feb. 14, 2022, both of which are incorporated by reference in their entireties herein.

TECHNICAL FIELD

This invention relates to a combination, such as a composition, and its therapeutic and prophylactic uses. In particular (although not exclusively) the invention relates to a new method to modulate or treat the microbiome of a canine animal through selectivity towards microorganisms. More particularly the invention relates to a new method to modulate or treat the microbiome of a canine animal through administration of a combination of milk proteins to the oral cavity.

BACKGROUND OF THE INVENTION

Animals host a very large variety of microorganisms, both commensals and pathogenic. Commensal microorganisms are those which live harmoniously with the host organism, utilising food or other benefits, without hurting it and often beneficially helping it. Oppositely, pathogenic microorganisms, including bacteria, fungi, or a virus, are organisms which, after invading the body, typically lead to infection and associated conditions or diseases. Occasionally, commensal microorganisms that are beneficial can take the opportunity to become pathogenic, in which case the commensals can be referred to as ‘opportunistic commensals’.

Collectively, the term microbiota describes the community of both commensals and pathogenic microorganisms that live on or in animals. The term microbiome is related to microbiota, but is often considered to describe the collective genomes of the microorganisms, rather than the microorganisms themselves. Throughout this specification, we will use the term microbiome, but this should be understood to encompass both the genetic and/or phenotypic diversity of the microorganisms.

The microbiome can be very diverse, and is present on a number of areas of the body including the skin, different areas of the gastrointestinal tract from the oral cavity or mouth through to the rectum, nasal cavities, ears, lungs and vagina. The different environments at each location leads to competition and adaptation of the microorganisms for survival. Additionally, in a healthy host, innate mechanisms help to selectively favour survival of commensals or health-giving microorganisms opposed to pathogenic microorganisms.

Research has established that a healthy microbiome is very important for metabolism of carbohydrates and proteins, development of the immune system, functioning of the epithelium, hormone production, vitamin production, pathogen protection and fat storage. (Hooper et al, 2004, Stappenbeck et al, 2004).

In the mouth, periodontal disease has been considered to be an infection with specific causative bacteria, however many periodontic bacteria are now considered to be permanent commensal bacteria rather than transient pathogens. The microbiota of the human oral cavity consists of a myriad of bacterial species that normally exist in commensal harmony with the host. For example, Porphyromonas gingivalis can be isolated from healthy individuals, but is involved in severe periodontal disease in some individuals with inflammation and loss of bone. In contrast to the human oral cavity, the canine oral cavity exhibits a different microbiome. For example, a low sucrose diet and other antagonistic Streptococcus app. are believed to lead to vastly reduced levels of Streptococcus mutans in canine animals in comparison to the oral cavity in humans. Porphyromonas gingivalis is generally considered to be pathogenic in canine oral cavities.

With knowledge of the importance of the microbiome, researchers have been investigating how to modulate it in order to maintain or improve overall health, defend or treat against infection and associated diseases or conditions.

The most commonly relied on approach is the use of antibiotics, which has been instrumental to modern medicine, both in terms of fighting infections that may otherwise kill a host, as well as allowing surgeries to be performed without major risk of subsequent infection and death. However, a major downfall of antibiotics, of course besides development of resistance, is that the antibiotics have little to no selectivity—such that the drug essentially kills all the microbiome, including the beneficial commensals. This is undesirable given the important functions of the microbiome as discussed previously.

Other approaches include the use of prebiotics and probiotics, which are thought to help modulate the microbiome. Prebiotics aim to provide optimal growing conditions for commensals. Probiotics include actual microorganisms with the aim of populating the body's microbiome with specific species with apparent beneficial outcomes. Synbiotics include a combination of pre- and probiotics. Although these approaches hold promise, there is little scientific evidence yet of the therapeutic effectiveness of modulating microbiomes for key desired health outcomes. Furthermore, although pre- and probiotics may help boost the system's defense system, it has little to no potency for treating an infection that has already manifested.

WO 2014/159659 describes a composition using a chelator and a base to selectively target pathogenic bacteria in dental diseases. A number of potential compounds are listed as potential enhancers, without any specific anti-microbial effect, but which enhances the effect of the chelator or base in some way. Yet, most of the enhancers were not investigated or shown to improve therapeutic effectiveness. Furthermore, there is no suggestion that the compositions used in WO 2014/159659 have selectivity to the microbiome outside the dental environment.

WO 2011022542 describes attempts to develop compositions with improved selectivity by relying on host-derived factors specific to each microbiome location, for instance in the mouth, skin, and airways. For instance, it discloses the use of salivary digestive products like maltose, maltotriose and dextrin to selectively modulate and promote commensals in the mouth. It also broadly suggests a range of other compounds with which may have additional benefits. There are seven example compositions provided, but without any analysis of whether these effectively work or impart any selectively towards commensals vs pathogenic microorganisms. Furthermore, WO 2011022542 teaches towards development of specific compositions with different active agents for each location of treatment. This can be seen as a complicated and undesirable system which requires very different components to be used as active ingredients for different locations.

A different line of scientific study has investigated the proteins and peptides of the innate defense system which are present throughout the body in all mammals. It is the first defense against the invasion of pathogens, is present in all parts of the body at all times and is independent of the systemic adaptive immune system. It is non-inflammatory because it does not invoke the production of cytokines and anti-inflammatory because it takes up free radicals.

The innate defense system is particularly important in the eyes, mouth and respiratory tract where there is high risk of the entry of harmful pathogens. These areas are protected by a constant flow of liquid (tears, saliva and mucous) containing a high concentration of the proteins, peptides and defensins of the innate system, and substrates such as thiocyanate, that are required by the peroxidase enzyme to produce hypothiocyanite.

Under this category, EP 0614352 describes a dentrifice composition that includes an oxidoreductase enzyme and its substrate in order to develop hydrogen peroxide once administered, thereby providing an antimicrobial effect from hypothiocyanite ion production. A number of oxidoreductase options are provided, including glucose oxidase as the preferred enzyme, together with its preferred substrate, glucose. As illustrated by Example E, other ingredients such as peroxidase may also be added in attempt to convert thiocyanate ions, in the presence of hydrogen peroxide, into hypothiocyanite ions. Although the compositions are shown to produce hydrogen peroxide, there is no evidence to show whether any compositions imparted any degree of selectivity towards pathogenic microorganisms instead of commensals. There is also no data to support whether addition of a peroxidase improves or imparts any selectivity. Furthermore, there are a wide number of synthetic excipients used, and there would appear a need to isolate or source each individual component before formulating the compositions.

As another example, U.S. Ser. No. 08/480,357 describes an approach to selectively target pathogenic microorganisms with an apparent lack of inhibition towards commensals. The document highlights that myeloperoxidase, in the presence of a peroxide generator (e.g. glucose oxidase) and halide such as Cl⁻ or Br⁻, provides some selectivity towards certain pathogens whilst apparently avoiding inhibition of specific commensals. However, there are wide variations between myeloperoxidase and other peroxidases tested in terms of selectivity and potency between pathogens, the binding data is often contradictory to the inhibitory results or suggestive of poor selectivity towards specific pathogens. Lactoperoxidase showed very poor binding selectivity in comparison (as shown in Table 13), suggestive of it having little to no inhibition or selectivity, albeit not actually tested by the authors. At best, U.S. Ser. No. 08/480,357 may motivate a reader to explore myeloperoxidase (or perhaps eosinophil peroxide as per the claimed invention in claim 1) usage together with a peroxide and halide to achieve the reported results. Regardless, this document does not report ideal selectivity results across a broad range of pathogenic bacteria together with a lack of inhibitory effects towards a broad range of commensals.

In the case of the mammary gland, all of the components of its innate defense system have been extensively studied, especially the major components from milk such as lactoferrin, lactoperoxidase and angiogenin (Ribonuclease). There are many publications describing activity of these proteins against bacteria, yeast, fungi and viruses. However none of these publications teach towards selectivity between commensals and pathogens, or the use of these components to modulate a microbiome.

In summary, new methods need to be developed to effectively modulate the microbiome without the harshness and lack of selectivity of antibiotics, and equally with greater potency than pre- and pro-biotics to selectively inhibit pathogenic microorganisms without a similar level of inhibition of the beneficial commensals. Furthermore, there is a need to address the shortcomings as discussed above in relation to WO 2011022542, WO 2014/159659, EP 0614352 and U.S. Ser. No. 08/480,357. Ideally, the approaches should rely on natural based compositions for consumer acceptance and avoidance of side effects. Preferably, the components are easy to source, extract and are shelf-stable.

It is an object of the present invention to address one or more of the foregoing problems or at least to provide the public with a useful choice.

Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only.

SUMMARY OF THE INVENTION

In one aspect the invention provides the use of a combination including lactoperoxidase and at least one other component, wherein the lactoperoxidase and at least one other component have an isoelectric point of or above substantially 6.8 and which are extracted from milk, to modulate a microbiome in a canine animal by selectively inhibiting growth or killing at least one pathogenic microorganism without a comparative inhibition of at least one commensal microorganism, by administering the lactoperoxidase and at least one other component to the oral cavity of the canine animal.

In one aspect the invention provides a method of modulating a microbiome in a canine animal by selectively inhibiting growth or killing at least one pathogenic microorganism without a comparative inhibition of at least one commensal microorganism, the method including the step of administering to the oral cavity of the canine animal a combination including lactoperoxidase and at least one other component, wherein the lactoperoxidase and at least one other component have an isoelectric point of or above substantially 6.8 and which are extracted from milk.

In one aspect the invention provides a method of treating or preventing a condition or disease in a canine animal that has at least a partial causative association with a microbiome in at least one location on or in the canine animal, the method including the step of administering to the oral cavity of the canine animal a combination including lactoperoxidase and at least one other component, wherein the lactoperoxidase and at least one other component have an isoelectric point of or above substantially 6.8 and which are extracted from milk.

Prefer the combination including lactoperoxidase and at least one other component is a composition including lactoperoxidase and the at least one other component.

Preferably the combination, such as the composition, includes lactoperoxidase, together with at least one or more of lactoferrin, angiogenin, and/or lysozyme-like protein, all having an isoelectric point of or above substantially 6.8 and which are extracted from milk.

Preferably the combination, such as the composition, includes lactoperoxidase, quiescin, jacalin-like protein, and angiogenin, all having an isoelectric point of or above substantially 6.8 and which are extracted from milk.

Preferably the combination, such as the composition, includes lactoperoxidase, lactoferrin, angiogenin, and lysozyme-like protein, quiescin, and jacalin-like protein, all having an isoelectric point of or above substantially 6.8 and which are extracted from milk.

In some embodiments the combination, such as the composition, includes cathlecidin-1 and/or serum amyloid A.

Preferably the combination, such as the composition, includes substantially all proteins isolated from milk which have an isoelectric point of or above substantially 6.8.

In summary, the inventors have discovered that the combinations, such as the compositions, as described herein have an unexpected selectivity towards inhibition of pathogenic microorganisms in the oral cavity of a canine animal compared to a considerably less inhibitory effect towards beneficial commensal microorganisms that are present in a healthy canine microbiome. The discovery presents itself towards new uses for modulating microbiomes, and/or preventing or treating associated conditions or diseases. Furthermore, the invention provides a significant advantage over previous methods of treatment such as broad spectrum antibiotics which do not have a high degree of specificity. The invention is also hugely beneficial in the sense that the combination, such as the composition, may be produced easily using known techniques and includes proteins derived from milk that have a wide canine acceptance and safety profile. Finally, the convenience of being able to isolate combinations of the milk proteins together during manufacture and storage also appears to be improving overall anti-microbial effect, and early indications suggest this also improves retention of the beneficial selectivity profile.

Preferably the combination, such as composition, of the invention is used to modulate the microbiome of the oral cavity and/or gut (which may refer to the whole or part of the gastrointestinal tract (esophagus, stomach, small intestine, and large intestine)) of the canine animal. Preferably the combination, such as composition, of the invention is used to modulate the microbiome of at least the oral cavity of the canine animal.

The canine oral cavity microbiome is widely divergent from that of humans (Dewhirst F E, Klein E A, Thompson E C, Blanton J M, Chen T, Milella L, et al. in (2012) The Canine Oral Microbiome. PLoS ONE 7(4): e36067). For example, S. mutans is found in the human oral microbiome and may be cariogenic in humans, but is not found in the canine oral microbiome (Káthia Santana Martins, Lorena Tirza de Assis Magalhães, Jeferson Geison de Almeida, Fábio Alessandro Pieri, “Antagonism of Bacteria from Dog Dental Plaque against Human Cariogenic Bacteria”, BioMed Research International, vol. 2018, Article ID 2780948, 6 pages, 2018. https://doi.org/10.1155/2018/2780948). By way of another example, Porphyromonas gingivalis was listed as a commensal organism in patent publication WO2017183996, however the organism has been implicated as a periodontal pathogen in the oral cavity of canines (Braz. J. Microbiol. 38 (1) March 2007). Several theories for this difference have been postulated including that “simple carbohydrates and sugars are not normally a major constituent of the canine diet and canine saliva has a pH of approximately 8.0 (WALTHAM, unpublished data 2011) which may be hostile to members of this aciduric genus” (Dewhirst et al.). Other reports suggest that halitosis (bad breath) in canine animals may represent the first clinical sign of periodontal disease, and that halitosis may be remedied by treatment of the periodontal disease with supragingival scaling and polishing to remove all dental deposits and tooth extraction where necessary (Culham, N. & Rawlings, J. M. American Society for Nutritional Sciences. J. Nutr. 128: 2715S-2716S, 1998).

Further studies into the diversity of the canine microbiome (particularly that of the nasal and oral cavities) have been reported by Bailie, W. E. et al. in Journal of Clinical Microbiology (1978) 7(2) 223-231, exhibiting a diverse range of microorganisms that may be common with humans or peculiar to canines.

Such differences may impact on the ability of previously known antimicrobial agents to successfully and beneficially modulate the canine oral microbiome.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the general elution profile of all the fractions from cation exchange. This represents all the protein peaks (as detected at 280 nm) that would be present in a single fraction eluted in a gradient from 80-100 mS. The main components in the cationic fraction are immunoglogulin, lactoperoxidase, lactoferrin, and a group of minor components that include angiogenin.

FIG. 2 shows the fractions separated on SDS-PAGE, and indicates the band that was excised for Mass Spectroscopy and identified as bovine angiogenin. The immunoglobulin fraction shows PIGR (76 kDa) as the predominant band, and the heavy (52 kDa) and light chains of immunoglobulin. The Lp fraction is mainly lactoperoxidase with a small amounts of heavy and light chains of immunoglobulin and angiogenin. The intermediate fraction has a prominent band of lactoperoxidase and lactoferrin (80 kDa) and a band at around 15 kDa that was identified by Mass Spectrometry as angiogenin, a band at approximately 13 kDa that was identified by Mass Spectrometry as jacalin-like. The Lf fraction is predominantly lactoferrin (80 kDa).

FIG. 3 shows a graph of pathogenic Escherichia coli inhibition using the cationic fraction alone, and with 40 ppm of sodium thiocyanate,

FIG. 4 shows a graph of pathogenic Streptococcus uberis inhibition using the cationic fraction alone, and with 75 ppm of sodium thiocyanate and 150 ppm of ascorbate,

FIG. 5 shows a graph of pathogenic Escherichia coli growth using various sub-fractions of the cationic fraction, a recombined cationic fraction and an unfractionated (whole) cationic fraction,

FIG. 6 shows a graph of pathogenic Staphylococcus aureus growth using various sub-fractions of the cationic fraction, a recombined cationic fraction and an unfractionated (whole) cationic fraction

FIG. 7 shows a photograph of an agar plate used to grow A. actinomycetemcomitans anaerobically, which was exposed to the combination of the invention at four dilutions.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “modulate” takes its normal meaning which is to exert a controlling influence on a subject. For example, the combination may modulate pathogenic bacteria by inhibiting the growth of those bacteria. By way of another example, the combination may modulate commensal bacteria by inhibiting the growth of other bacteria, thus facilitating a suitable growing environment for those commensal bacteria. By way of another example, the combination may modulate commensal bacteria by inhibiting the growth of other bacteria more than the combination might inhibit the growth of the commensal bacteria.

As used herein the term “selectivity” refers to a difference in inhibitory activity towards commensal bacteria and against pathogenic bacteria and/or opportunistic pathogenic bacteria. Conveniently the level of selectivity may be represented numerically by comparing suitable quantified levels of inhibition, such as minimum inhibitory concentrations (MIC), such as MIC, MIC₅₀ or MIC₉₀ values. The comparison is typically made between two different species, but may also be made between strains within the same species. The comparison may be represented as a ratio of:

• • (the inhibitory activity towards a commensal bacterial species) to (the inhibitory ratio against a pathogenic bacterial species); or
  • (the inhibitory activity towards a commensal bacterial species) to (the inhibitory ratio against an opportunistic pathogenic bacterial species).

The level of selectivity may be low, medium or high, and may be quantified as being greater than or equal to 1.1, 1.5, 2, 5, 50, 100, 200 or 300.

As used herein the terms “inhibit”, “inhibition”, “inhibitory”, particularly with respect to bacterial growth, refer to a decrease in the rate of growth of the bacterial species with reference to the uninhibited rate of growth of the bacterial species. Typically bacterial growth can be measured by counting the change in the number of cells as a function of time, although other methods such as medium digestion, metabolite production, etc are envisaged. In some embodiments, the degree of inhibition is determined by measuring the difference in rate of growth of a population of a bacterial species as a function of time as compared to a different population of the bacterial species grown in the same conditions without the inhibitor, such as the combination of the present invention.

As used herein the term “oral cavity” in relation to a canine animal refers to the space and associated structures bound laterally and rostrally by the lips and cheeks, dorsally by the hard palate, and ventrally by the tongue and underlying mucosa. As used herein the oral cavity includes the surfaces of teeth and gums.

Diseases and/or conditions of the oral cavity include periodontal disease and dental caries. Periodontal disease is the most common oral canine disease and results from a complex interplay between plaque bacteria, the host and environmental factors. Periodontal disease affects canine teeth and the surrounding structures (the gums and bone). Periodontitis can result in gum infections, bone loss and, if left untreated over time, the loss of teeth and other serious health problems. Dental caries initiate from acidic demineralization of dental enamel which may be due to cariogenic bacteria releasing acids onto the tooth surface. If left untreated acids released by bacteria then demineralise dentine, permitting bacteria to invade deeper, until significant loss of structure of the crown is encountered.

Preferred Features of the Combination (for Example the Cationic Fraction of Milk)

Throughout this specification, use of the term ‘cationic fraction’ should be taken as meaning a fraction or isolated components from a milk, being cationic components that bind to cation exchange media, and include any component of milk which has an isoelectric point of or above substantially 6.8.

Throughout this specification, the term “commensal” should be taken as meaning an organism that is normally harmless to the host, and can provide beneficial effects to the host.

The inventors found that some or all the proteins in the cationic fraction isolated from milk are collectively working together to somehow induce highly beneficial selectively towards numerous pathogenic microorganisms without a comparative level of inhibition of commensals in the canine oral cavity. Initial trials have indicated that selectively is synergistically enhanced if the proteins of the cationic fraction of milk are retained together, rather than combined.

As used herein, the term “synergistic” means that the effect achieved with the compositions and combinations of the invention is greater than the sum of the effects that result from using the individual components as a monotherapy. Advantageously, such synergy provides greater efficacy at the same doses, and provides an effect where otherwise there would be no discernible effect.

It should be understood that the particular method of combining the proteins in the composition together, which appear to provide advantageous selectivity, should not be considered to be a limitation to the invention at hand. For instance, with knowledge of the selectivity results observed herein and a clear understanding of what proteins are in the cationic fraction, a person skilled in the art could potentially prepare a combination of proteins from different sources, or even potentially synthetically engineer each protein and combine them as appropriate. However, the ability to separate and elute a cationic fraction using chromatographic methods represents a convenient way to prepare the combination(s) of the invention as a composition, and also provides a delicate mechanism to keep the proteins in their innate environment to avoid loss of protein function or inter-engagement with the other milk proteins, and to promote any form of synergism that appears to be at play between the proteins.

The proteins used in the combination, such as the composition, may be isolated or extracted from one or more sources of milk; such as non-canine milk; such as bovine milk, sheep milk, goat milk, buffalo milk, camel milk, human milk and the like; such as bovine milk, sheep milk, goat milk, buffalo milk, and/or camel milk. The major and minor proteins found in bovine milk (used for this preliminary study) are also found in other sources of milk, with very similar isoelectric points in each case. Additionally, the term milk should be taken to include whole milk, skim milk or whey.

Therefore, based on the closely related proteins found in such milk sources, one would expect such proteins to synergistically work together in combination to provide a similar selectivity response, and could be conveniently extracted and stored together as a cationic fraction isolated from any given milk source.

In one preferred embodiment the cationic fraction may a molecular weight distribution of 3,000-80,000 Daltons by SDS-PAGE.

This protein size distribution range encompasses the size of the proteins observed within the cationic fractions (and sub-fractions) of milk.

The most prevalent proteins in the cationic fraction and proteins in preferred embodiments of the present invention are lactoferrin, angiogenin and lactoperoxidase. The relative amounts do vary a lot in milk. Typically, the cationic fraction (and therefore potentially the resulting combination, such as the composition,) may include lactoferrin in the range between 20-70% w/w and lactoperoxidase in the range between 5-40% w/w. The inventors believe these proteins may be primarily responsible for the impressive, yet unexpected selectivity towards pathogens in favour of promoting the commensals in the microbiome.

However, there also a wide number of additional proteins in milk which may be isolated as part of the cationic fractions and combinations, such as a compositions, studied by the inventors, many of which may also be contributing towards the beneficial selectivity observed across a wide number of pathogens vs commensals.

Without limitation, the proteins found in the cationic fraction of milk, and also considered to be relevant to the invention at hand, are discussed in more detail below. It should be appreciated that although many of these proteins are thought to be associated with an innate immune response and/or impart some level of biocidal activity, it has never been elucidated that the proteins from milk may work together synergistically to provide selectivity towards microorganisms to help modulate a microbiome environment.

Lactoperoxidase

Lactoperoxidase (Lp) is a protein present in the mammary gland secretion and many other exocrine secretions of mammals.

The Lactoperoxidase system consists of three components—Lp, thiocyanate and hydrogen peroxide, which are all present in fresh milk. Lp catalyses the oxidation of thiocyanate by peroxide and generates intermediate products (hypothiocyanite (OSCN⁻)), with antibacterial properties. Thiocyanate is present in the mammary, salivary and thyroid glands and their secretions, in synovial, cerebral, cervical and spinal fluids, in lymph and plasma, and in organs such as stomach and kidney. Hydrogen peroxide, the third component of the Lactoperoxidase system is not normally detected in milk, but is present during infection.

The Lactoperoxidase system has bacteriostatic or bactericidal activity on a variety of susceptible microorganisms including bacteria, fungi and viruses associated with mastitis.

The inventors previously identified through a major R&D product pre-2006 (the subject of NZ Patent 547849) that the cationic fraction, which includes lactoperoxidase and other preferred proteins isolated from milk, was able to be used effectively to treat mastitis by attacking the causative microorganisms in the mammary gland after administration. This major breakthrough over 10 years ago was important because it provided a treatment which cleverly engaged from the innate immune response of the mammalian mammary gland environment, and utilised endogenously found proteins produced by the actual mammary gland. Furthermore, the composition's efficacy did not rely on conventional antibiotic treatment, which in the dairy industry was an important feat as it avoided issues with increased antibiotic resistance, long withholding periods and addressed a growing consumer desire for natural alternatives to antibiotic treatments. The inventors also envisaged at the time that the same composition would also be useful to treat infections on other areas on the body besides mastitis.

However, the inventors have now only recently identified the proteins found in this cationic fraction have an important further therapeutic and commercial characteristic, that being high selectivity toward pathogenic microorganisms yet with a dramatic low inhibition of beneficial commensals. This opens up an entirely new application of the milk proteins to improve microbiomes present on or in the body of humans or other animals.

Furthermore, it overcomes the problems associated with administering antibiotics which are not selective towards pathogens, and therefore destroys the entire microbiome, including both pathogens and commensals. Prebiotics or probiotics act to more conservatively boost the commensals but without any direct inhibitive function. However, the present invention has a direct inhibition effect on pathogens, making it a more potent tool to combat infections, or conditions which have a poorly functioning microbiome overridden with pathogenic microorganisms.

In fact, this newly discovered selectivity opens the door to potentially combining the selectively antimicrobial milk-derived components with prebiotics or probiotics in order to further enhance the balance of the native microbiome, whilst simultaneously exogenously adding to the natural commensal activity. This is a functional advantage of the present invention that would not otherwise have been possible with prebiotics or probiotics alone.

Quite contrary to the present invention, U.S. Pat. No. 5,888,505 describes that different forms of peroxidase have a wide variety and specificity towards targeting pathogenic microorganisms. The results suggested that lactoperoxidase was effectively providing no or very little selectivity between the different microorganisms compared to the other forms of peroxidases. Contrary to this, the inventors have now discovered that the cationic fraction in milk, which relies exclusively on lactoperoxidase action (instead of other peroxide forms) yet together with other protein(s) isolated from milk with a pl above 6.8, performs remarkably well, and in fact appears to significantly outperform the specificity and broad spectrum effectiveness seen in U.S. Pat. No. 5,888,505. Furthermore, the present invention does not require peroxide or halide addition for therapeutic effectiveness (although either may be included if the treatment site is devoid of natural substrates like peroxide/halide), and does not require high concentrations of lactoperoxidase in order to achieve a desirable knockdown effect. Additionally, there is reasonably good consumer acceptance of lactoperoxidase in commercial products. Further to this, milk proteins are stable and can be sourced cheaply.

U.S. Pat. No. 6,544,498 discloses the extraction by gradient elution of a basic protein fraction which has an isoelectric point between 7.5 and 11 and a molecular weight distribution of 3,000 to 80,000 Daltons, with the main components being lactoperoxidase and lactoferrin. U.S. Pat. No. 6,544,498 argues that the inventiveness of their application is based on the fraction curbing the decrease in alveolar bone and shows experimental data supporting this. There is no indication that the composition in U.S. Pat. No. 6,544,498 was identified to have selectivity towards pathogenic bacteria without a similar inhibition towards commensals, nor was there any discussion that selectivity could be enhanced dramatically by combining many, if not all, of the suite of proteins as described in the present invention with milk proteins with a pl above 6.8. In fact a number of subsequent publications confirm that the protein fraction in U.S. Pat. No. 6,544,498 acts to curb the decrease in alveolar bone by promoting osteoblast proliferation (see for example: U.S. Pat. No. 8,647,619; Aoe, S., et al. A controlled trial of the effect of milk basic protein (MBP) supplementation on bone metabolism in healthy menopausal women. Osteoporosis International 2005; 16:2123-8; Yamamura, J., et al. Milk basic protein (MBP) increases radial bone mineral density in healthy adult women. Bioscience, Biotechnology, and Biochemistry 2002; 66(3):702-4; Dorit Naot; Andrew Grey; Ian R. Reid; Jillian Cornish Lactoferrin—A Novel Bone Growth Factor Clin Med Res. 2005 May; 3(2): 93-101) rather than by any antibacterial activity, let alone selective antibacterial activity. Therefore, U.S. Pat. No. 6,544,498 does not teach towards the invention.

Lactoferrin

Lactoferrin (Lf) is a glycoprotein which is present in mammary gland secretion and many other exocrine secretions of mammals. Lf is secreted predominately by surface epithelia into the mucosal environment. Lactoferrin is a multifunctional protein that has antibacterial, antifungal, antiviral, antitumour, anti-inflammatory, and immunoregulatory properties. Therefore, the inventors expect that Lactoferrin is contributing to the anti-microbial effects of the combination, such as the composition, but more importantly is somehow, in combination with the other protein(s) in the combination, helping to impart an intricate level of selectivity towards pathogenic microorganisms yet with very low MIC levels towards commensals.

Lf is produced at high levels in nasal and tracheal passages, and in gastric, genital and ophthalmic secretions. Lf is also produced at high levels in neutrophils where it is stored in secondary granules and released during inflammation.

The mechanism by which Lf inhibits microbial growth has not been fully elucidated. Its antimicrobial and anti-inflammatory effects are believed to be as a result of a number of different actions or functions of Lf.

The highly basic N terminal region of bovine lactoferrin is thought to be essential for antimicrobial activity. The 25 N-terminal amino acids may be removed by proteases to form lactoferricin (Lfcin). These proteases may be naturally occurring in milk or serum, and many microorganisms produce proteases. Lfcin is up to a 1000 fold more effective against some microorganisms than intact lactoferrin. Lfcin has been shown to inhibit a diverse range of microorganisms such as gram-negative bacteria, gram-positive bacteria, yeast, filamentous fungi, and parasitic protozoa, including some antibiotic-resistant pathogens. Therefore, it is plausible that lactoferricin may be added to the combination, such as the composition, replace lactoferrin, and/or be a natural degradation product of lactoferrin in the combination of the present invention due to proteolytic action.

Lf binds to lipopolysaccharide. When Gram-negative bacteria are killed by the natural defense system of the animal or by antimicrobial agents the release of lipopolysaccharide from the cell walls of the bacteria provokes an inflammatory response. One of the primary actions of Lf therefore is to bind the LPS and prevent the inflammatory response. Lf also displays an immunomodulatory role by binding with high affinity to bacterial endotoxin, thus protecting against endotoxin lethal shock.

Lf is also an iron binding glycoprotein. Most microorganisms need iron for growth and therefore Lf has the potential to inhibit the growth of bacteria and even kill them by depriving them of iron. The effectiveness of the anti-bacterial activity of Lf depends on the iron requirement of the organism, availability of exogenous iron, and the concentration and degree of iron saturation of Lf.

Current commercial applications of bovine Lf include infant formulas, fermented milks, nutritional iron supplements, chewing gums, immune-enhancing nutraceuticals, cosmetic formulas and feed and pet care supplements. Therefore, it is advantageous to note that there is general consumer acceptance, and food safety regulations for use of Lactoferrin in the combination, such as the composition.

Angiogenin-Ribonuclease

Angiogenin-Ribonuclease belongs to the ribonuclease superfamily have been identified in milk, and is known to have some anti-viral and anti-microbial activity. Therefore, the inventors expect that Angiogenin-Ribonuclease is contributing to the anti-microbial effects of the combination, but more importantly is somehow (in combination with the other protein(s) in the combination) helping to impart an intricate level of selectivity towards pathogenic microorganisms yet with very low MIC levels towards commensals.

Lysozyme-Like Proteins, Such as Chitinase-Like Protein (CLP-1) or Lysosomal Alpha Mannosidase (LAM)

The combination preferably includes lysozyme-like protein, such as chitinase-like protein (CLP-1) or lysosomal alpha mannosidase (LAM). Lysozyme-like proteins (such as CLP-1 or LAM) have cell lysing activity and thereby are thought to enhance antimicrobial activity through their lysozyme-like effects.

In a preferred embodiment, the combination (such as the cationic fraction) may also include quiescin and/or jacalin-like protein.

Other milk proteins that may be included within the combination to improve its effectiveness (either through imparting selectivity, or some other form of indirectly modulation of the protein(s) functionality) include:

• • cathelicidin 1;
  • N-acetyl glucosaminidase;
  • serum amyloid A;
  • β Defensin;
  • Peptidoglycan recognition protein;
  • Xanthine dehydrogenase;
  • Immunoglobulin(s) IgA, IgD, IgG, IgM, IgA, and/or IgE;
  • Growth factors EGF, IGF 1, TGF B1 and TGF B2.

Without wishing to be bound by theory it is believed that the use of cathlecidin 1 and/or serum amyloid A in the composition of the invention, both of which have an isoelectric point (pl) of less than 7.5, may be responsible in part for advantageous selectivity.

Immunoglobulins are important components of milk and provide passive protection to the suckling young. Although they are not strongly cationic some immunoglobulins, IgG, IgM, IgA and polymeric immunoglobulin receptor (PIGR) are captured by cation exchange. Immunoglobulins are important in the first line of defense against foreign invaders. Immunoglobulins bind to microorganisms and thus opsonise them so that they are more easily recognized by phagocytic cells. It is plausible, therefore, that they may also have some effect on the observed selectivity in the present invention, and may be working synergistically with other proteins in the cationic fraction.

It is anticipated that the combination (such as the cationic fraction) isolated from milk may also include small amounts of a number of growth factors; although these growth factors may be present at low levels, their action can be potent in stimulating cell repair. These growth factors may include for example: EGF, IGF 1, TGF B1 and TGF B2.

Interestingly, Smolenski et al. (2007) reported on the identity and significant number of minor proteins in bovine milk by Mass Spectrometry (MS) and, in particular, identified a significant number of milk proteins that are involved in host defense. Yet, Smolenski in no way mentions or suggests any selectivity for any of the individual proteins, or combination of proteins despite mentioning that individual proteins have anti-microbial activity. The results are shown in Table 1, which we have adapted to show, in bold, some of the proteins which correspond to those preferably incorporated into the combination of the present invention (and which were isolated via the cationic fraction in milk and shown to have high selectivity according to the present invention). It should be noted that Smolenski et al. (2007) used SDS-PAGE methods that do not disclose the detection of the proteins identified in the combinations (such as compositions including the cationic fraction) used in the present invention (e.g. angiogenin, jacalin-like protein, quiescin, PIGR and the growth factors).

Table 1. Host defense-related minor proteins identified from milk, showing some of those that may be extracted as part of the cationic fraction (bold) (reproduced from Smolenski et al., 2007)

TABLE 1
Minor proteins identified in bovine milk.

ACC Number	Protein Name	Function	pI

NP_777250	cathelicidin 1 (Bactenecin 1)	antimicrobial properties	6.8*
AAB64304	chitinase-like protein 1	eosinophil chemotactic properties	8.8
	(CLP-1)
Q290092	endoplasmin precursor	participates in the assembly of	4.7
	(GRP94/GP96)	antibody molecules and signaling
		molecule for polymorphonuclear
		neutrophils
NP_776758	glucose regulated protein	regulates signaling by interacting with	unknown
	58 kDa	stat3
NP_776770	heat shock 70 kDa protein 8	activated through proinflammatory	5.4
		response mechanisms enhancing MMP-
		9 expression in monocytic cells
NP_071705	heat shock 70 kDa protein 5	upregulation in macrophages upon IL-4	unknown
	(glucose-regulated protein)	stimulation
AAA18337	heat shock protein 27	inhibitor of neutrophil apoptosis	5.98*
BAA32525	heat shock protein 70 kDa	stress response (refolding and	5.68*
	protein 1A	degradation of denatured proteins)
AAC98391	immunoglobulin IgA	antigen recognition	X1
AAN07166	immunoglobulin IgD	antigen recognition	X1
AAB37381	immunoglobulin IgG	antigen recognition	X1
AAN60017	immunoglobulin IgM	antigen recognition	X1
AAQ88452	IRTA2	B-cell immunoglobulin super-family	unknown
		receptor
AAA30617	lactoferrin	iron binding and antimicrobial peptide	8.67*
		“lactoferricin”
NP_776358	lactoperoxidase	oxidative peroxidase activity	8.327*
BAA07085	lymphocyte cytosolic	regulation of neutrophil integrin	5.21*
	protein 1 (65K macrophage	function
	protein/L-plastin)
P21758	macrophage scavenger	mediate the binding, internalization	5.7*
		and processing of negatively charged
		macromolecules
AAA36383	nucleobindin 1	promotes production of DNA-specific	5.05*
		antibodies
NP_776998	peptidoglycan recognition	innate immunity pattern recognition	9.38*
	protein	molecule
XP_611685	S100 calcium binding	associated with S100A8 and implicated	6.29*
	protein A9 (calgranulin B)	in inflammatory response
XP_593653	S100 calcium binding	upregulation associated with	6.7
	protein A11 (calgizzarin)	proinflammatory response
NP_777076	S100 calcium binding	antimicrobial peptide “calcitermin”	5.9
	protein A12 (calgranulin C)
P42819	serum amyloid A protein	involved in acute phase cytokine	6.94
		signaling
CAA67117	xanthine dehydrogenase	superoxide anion, hydrogen oxide and	8.0
		peroxynitrite production
1Immunoglobulins typically have isoelectric points the range of 5.0-9.5. As such, not all bind to the cationic exchange resin.
*The isoelectric points of these proteins have been calculated based on the expected protein structure. (Swiss Prot/TrEMBL, www.expasy.org).

Some of the cationic fraction components (e.g lactoferrin, angiogenin) may also have minor variants, such as variations in amino acid sequence or in degree and type of glycosylation, these minor variants, and their presence in the cationic fraction should also be taken as being covered by the present application.

The combination of the invention, such as the composition, may be administered to the oral cavity of the canine animal in the form of a liquid, cream, gel, paste, powder, capsule, lozenge, tablet, suppository, bolus, injectable solution and so forth. The combination may be administered as part of a food, such as a chew, or a drink.

A dog chew is a particularly preferred administration form for administering the combination of the invention to a canine animal. In some embodiments, it may be preferable for the combination of the invention to be administered on a regular or semi-regular basis to the canine animal. To that end, the owner of the canine animal will generally find it more convenient to incorporate the administration routine into the normal routine of the canine animal, and as such including the combination as part of a food (such as a chew) achieves the dual function of providing nutrition and also modulating the microbiome of the oral cavity of the canine animal. However by administering the combination as part of a foodstuff, it is inevitable that at least some of the combination will be consumed into the gut of the animal, which is likely to have a different microbiome to the oral cavity. Advantageously it has been shown that the beneficial effects of inhibiting growth of pathogenic microorganisms in the oral cavity of the canine animal (such as P. gingivalis and A. actinomycetemcomitans) that have been achieved using the combination of the invention are not offset by deleterious effects to the gut microbiome. The combination of the present invention has been demonstrated not to inhibit beneficial gut microorganisms (such as Lactobacillus acidophilus which is given to humans and dogs as a probiotic, primarily to improve gut health).

Dog chews may include at least one of: binding agents; softening agents (which may be an anti-sticking agent); an anti-caking agent or lubricant; a humectant or wetting agent flavourings; vitamins; and colors to enhance the manufacturability, texture and appearance of the product. The components will generally be at least food grade quality.

Examples of humectants are glycerol and propylene glycol. Examples of wetting agents are cetyl alcohol and glycerol monostearate.

Examples of softening agents are polysaccharides and fiber, which may be also provided as a filler or as a bulking agent and to provide or maintain porosity in the edible soft chew. Examples of fibers may be derived from fruits, grains, legumes, vegetables or seeds, or provided in forms such as wood fiber, paper fiber or cellulose fiber such as powdered cellulose fiber.

Where present, the binder may be a sticky substance, but will preferably give the edible soft chew product a food-like texture. Examples include molasses, corn syrup, peanut butter, a starch (such as potato starch, tapioca Starch or corn starch), honey, maple syrup and sugars. Preferred binders for use in dog chews of the invention are starches.

The combination of the invention, such as the composition, may include at least one or more of the following: carriers, buffers, preservatives, excipients or other pharmaceutically acceptable components required to ensure the cationic fraction is in a form that is easily dispensed, used and is efficient for the purpose of selectively supporting the microbiome.

Other creative means for administering the combination of the invention, such as the composition, to the oral cavity of the canine animal include through the use of a non-edible object in which the combination is impregnated/infused/absorbed/adsorbed. By way of example only, a solution the combination, such as the composition, may be impregnated in a solid object, such as a dog play toy, such as a rope. As the dog plays with the toy, and bites the toy, the combination will release from the toy, potentially aided by saliva from the dog's mouth. This release may be rapid, or sustained. In some examples, the release may be sustained over a period of several minutes or more, such as about 10 minutes, or about 20 minutes. The combination of the invention, such as the composition, may be impregnated/infused/absorbed/adsorbed by soaking the solid object, spraying the solid object, and/or injecting the solid object with a solution of the combination of the invention. Such a solution may be quite dilute, such as 0.1-5% w/v, such as 0.33-2% w/v. In trials conducted to date, such a method of administration to the oral cavity of a canine animal may allow for up to 100% recovery of the combination after repeated extraction under simulated conditions of a dog chewing on the solid object (rope). Spraying the solid toy led to the fastest release of the combination. Soaking led to the slowest release. The user of the technology may wish to select the method of impregnation/infusion/absorption/adsorption according to the desired release profile.

In one embodiment the combination of the invention, such as the composition, may also include at least one component which is capable of controlling the time release of the combination. This may be used to effectively to extend the release of the therapeutic effect over an extended period of time. Known components which could be used for this purpose would be well known to one skilled in the art.

The combination, such as the composition, may also include one or more of the following:

• • 1. a peroxidase substrate,
  • 2. hydrogen peroxide or a source of hydrogen peroxide, such as ascorbate or ascorbic acid;
  • 3. a cell-lysing substance capable of fully, or partially lysing cell walls (such as detergents like monoglyceride or monolauryl glycerol [monolaurin]).

The peroxidase substrate may be any substrate or compound on which lactoperoxidase or any other peroxidase enzymes may act. In one preferred embodiment the peroxidase substrate may be thiocyanate.

In one particular preferred embodiment the peroxidase substrate may be potassium or sodium thiocyanate. Alternatively any other thiocyanate which can act as a peroxidase substrate may be utilized.

In a preferred embodiment the minimum concentration of peroxidase substrate is 20 ppm (when the peroxidase substrate is sodium thiocyanate), 20 ppm (when the source of hydrogen peroxide is ascorbate) and 5 ppm (when the cell lysing agent is monolauryl glycerol) (as shown in vitro).

Ascorbate and ascorbic acid have been shown in previous publications to be good substrates for peroxidase enzymes. This is a preferred source of hydrogen peroxide as it is stable—unlike peroxide itself. Hydrogen peroxide is also a substrate of peroxidase enzymes.

Therapeutic Uses

Preferably (yet without limitation), the pathogenic and/or commensal microorganisms are selected from the group consisting of gram-positive bacteria, gram-negative bacteria, aerobic bacteria, anaerobic bacteria, fungi, yeasts and/or viruses.

The present inventors have discovered that the combination of the invention shows a clear selectivity to inhibit a wide selection of pathogenic microorganisms (or which can become pathogenic if the microbiome is compromised) including:

• • Propionibacterium acnes,
  • Streptococcus pyogenes,
  • Candida albicans,
  • Trichophyton mentagrophytes,
  • Trichophyton rubrum,
  • Malazzezia furfur,
  • Escherichia coli,
  • Streptococcus mutans,
  • Staphylococcus aureus,
  • Actinobacillus actinomycetemcomitans.

These pathogenic microorganisms are implicated in infections on the skin, hair, nails, gut, nose, ears, oral cavity, vagina, anterior urethra, lungs and/or any other areas of the body that has a surface that is either accessible to external gases, liquids, foods, etc. or is isolated from internal systems via a blood barrier, such as the mammary gland, which exemplifies the broad therapeutic uses of the combination.

In patent publication WO2017183996, Porphyromonas gingivalis was listed as a commensal organism. While this may be generally true, the canine oral cavity is an exception and the organism has been implicated as a periodontal pathogen in canines (Braz. J. Microbiol. 38 (1) March 2007). That same reference also implicates Actinobacillus actinomycetemcomitans.

It is also believed that the combination of the invention may inhibit Porphyromonas cangingivalis, which can cause periodontitis in dogs (Ruparell, A., Inui, T., Staunton, R., Wallis, C., Deusch, O., & Holcombe, L. J. (2020). The canine oral microbiome: variation in bacterial populations across different niches. BMC Microbiology, 20(1), 42. https://doi.org/10.1186/s12866-020-1704-3).

In conjunction, the initial trials conducted by the inventors have shown that the combinations show a comparatively low level of inhibition across a wide range of commensal microorganisms, which are outlined in greater detail below. For instance, the MIC (mg/ml) for commensal microorganisms may be 20-100 fold (Table 4) higher compared to the MIC for most pathogens. This means the combination has low inhibitory effectiveness towards commensals, such that they can proliferate and populate the microbiome, whilst the pathogens at the site of infection are attacked by the combination.

It is believed that the combinations would show a comparatively low level of inhibition against Streptococcus minor, which has been recently described, and is found in the tonsils and gastrointestinal tract of dogs. This has been isolated from dog bite wounds in people but is not believed to cause disease.

The inventors also expect that the combination may have the ability to selectively modulate opportunistic commensals, which although imparting benefits to the host in a healthy environment, may cause infection or harm if the microbiome is either weakened, or if the commensal enters through a barrier such as the skin or gastrointestinal lining, for instance due to an injury. In such a case, the commensal may become pathogenic, and the combination may have the ability to selectively modulate the microbiome, thereby lessening the chance of or the severity of the infection. There are many examples of commensals which may become opportunistic.

The synergistic therapeutic effectiveness of the proteins in combination (emphasised by comparison to the results in U.S. Pat. No. 5,888,505) is also cleverly making use of the easy ability to isolate the proteins together in a delicate manner to preserve their native interactions with other proteins, and to preserve stability of the combination before consumption.

Areas to be Treated

The present invention is specifically directed to administering the combination to the oral cavity of a canine animal. Whilst not limiting, the canine will generally be a domestic dog. More specifically the present invention is directed to selectively inhibit growth or kill at least one pathogenic microorganism in the oral cavity of a canine animal without a comparative inhibition of at least one commensal microorganism. The commensal microorganism may or may not be in the oral cavity of the canine animal. The commensal microorganism may be in the gut of the canine animal. It will be appreciated that administering a combination to the oral cavity of a canine animal to inhibit growth or kill at least one pathogenic microorganism in the oral cavity may ultimately lead to at least part of the combination being located in another part of the animal, such as the stomach or further along the digestive tract. The present invention contemplates the commensal microorganism being located in one or more of those other parts. For instance, the combination of the invention may be used to inhibit growth or kill P. gingivalis and/or A. actinomycetemcomitans without a comparative inhibition of a gut commensal such as Lactobacillus acidophilus.

The canine oral microbiome is widely divergent from that of humans (Dewhirst F E, Klein E A, Thompson E C, Blanton J M, Chen T, Milella L, et al. in (2012) The Canine Oral Microbiome. PLoS ONE 7(4): e36067). For example, S. mutans is found in the human oral microbiome and may be cariogenic in humans, but is not found in the canine oral microbiome (Káthia Santana Martins, Lorena Tirza de Assis Magalhães, Jeferson Geison de Almeida, Fábio Alessandro Pieri, “Antagonism of Bacteria from Dog Dental Plaque against Human Cariogenic Bacteria”, BioMed Research International, vol. 2018, Article ID 2780948, 6 pages, 2018. https:doi.org/10.1155/2018/2780948). Several theories for this difference have been postulated including that “simple carbohydrates and sugars are not normally a major constituent of the canine diet and canine saliva has a pH of approximately 8.0 (WALTHAM, unpublished data 2011) which may be hostile to members of this aciduric genus” (Dewhirst et al.). Such differences may impact on the ability of previously known antimicrobial agents to successfully and beneficially modulate the canine oral microbiome.

The combination of the invention may allow for separate, sequential or simultaneous administration of the lactoperoxidase and the at least one other component extract as hereinbefore described. The combination may be provided in the form of a composition, in which the lactoperoxidase and the at least one other component are in intimate admixture.

Typically a therapeutically effective amount of the combination of the invention will be administered. The term “therapeutically effective amount” refers to that amount which is sufficient to effect treatment, as defined below, when administered to canine animal in need of such treatment. The therapeutically effective amount will vary depending on the subject and nature of the disease being treated, the severity of the infection and the manner of administration, and may be determined routinely by one of ordinary skill in the art.

The terms “treatment” and “treating” as used herein cover any treatment of an infection in a canine animal, and includes: (i) inhibiting the infection; (ii) relieving the infection; or (iii) relieving the conditions caused by the infection, eg symptoms of the infection.

The terms “prevention” and preventing” as used herein cover the prevention or prophylaxis of an infection in a canine animal and includes preventing the infection from occurring in a a canine animal which may be predisposed to the infection but has not yet been diagnosed with the infection.

In some embodiments the combination of the present invention is formulated for topical administration to the oral cavity. Preferably, the combination of the invention will be applied topically such as in a paste, cream, lotion, gel or via an impregnated dressing or impregnated mask. The term “topical”, as used herein, may refer to a combination meant for application to mucosal tissue (such as gum).

Combination Products

The applicant envisages that the combination of the present invention may include other compounds or material which is known or thought to promote or enhance the microbiome. For instance, the combination may include prebiotics or probiotics. It is possible this may enhance the selectivity profiles even further.

Methods of Manufacture and Storage

Interestingly, the applicant discovered in previous studies that the inhibitory effects against the pathogen Streptococcus uberis (in the context of mastitis) diminished as the cationic bioactive fractions became more purified. This was contrary to common thinking as it is commonly understood that the purer a component is, the more effective it will be. At the time, this led to the hypothesis, which was subsequently tested that the ‘total cationic fraction’ of the present invention could be used as a successful naturally-derived inhibitory product. Additionally, previous research from this Applicant found that a combination of milk proteins (i.e. the cationic milk fraction with a pl above 6.8) induces a powerful anti-inflammatory action. Yet, it was not envisaged that this phenomenon would also have implications for beneficial selectivity towards pathogenic microorganisms vs commensals.

It should be appreciated that the term milk may include any raw (or unprocessed) milk. This is taken to include raw milk which has been chilled, incubated, or stored, at either a chilled or ambient temperature.

In one preferred embodiment the cationic fraction may be extracted from bovine milk.

However, this should not be seen as limiting, as the cationic fraction may also be extracted from other mammalian species, including, but not limited to sheep, goats, buffalo, camels and humans.

In one embodiment the proportions of the different cationic components within the cationic fraction may be as extracted, or concentrated.

However, this should not be seen as limiting, as it may be desirable to alter or control the ratio of at least one, or a number of components respectively. It should be appreciated that any such alteration in the proportions of the cationic fraction components are covered by this disclosure.

In one preferred embodiment the cationic fraction may be extracted “on-farm”, during or directly after the milking process. This may be advantageous as some of the components may be lost, damaged or denatured during subsequent handling, storage, fat removal, or other processing steps.

Many methods may be used to prepare a combination, such as a composition, as described according to the present invention. However, cationic exchange is considered to be a preferred method of manufacture, as will discussed in further detail below.

Preferably, the method includes extracting preferred proteins from milk, including the steps of:

• • a) passing milk through an extraction material, and
  • b) eluting a cationic fraction of the bound milk components having a pl above 6.8.

In a preferred embodiment the extraction material may be a cation exchange material. This may either be in the form of resin, expanded bed resin, magnetic beads, membrane or other suitable form for large scale extraction.

In a preferred embodiment the cation exchange material may be any material that has sufficient mechanical strength to resist high pressures and maintain high flow rates.

In a preferred embodiment the cation exchange resin may have a mean particle size in excess of 100 μm. Resins in larger bead form have been developed for use with viscous feed streams because they do not pack as closely as smaller beads therefore there are wider channels so that there is not excessive back-pressure.

Examples of suitable cation exchange resins are SP-Sepharose Big Beads, SP-Sepharose Fast Flow, SP-Toyopearl and S-Ceramic HyperD.

One example of an extraction and purification process is as follows:

Lactoferrin binds firmly to cation exchange and is the last major protein to elute in a salt gradient. Therefore a single step elution with 1M salt (80 mS-100 mS) elutes all proteins and peptides in a single fraction (cationic fraction). Elution with 80-100 mS salt following a prior 40 mS elution will yield a fraction that is primarily lactoferrin.

After lactoferrin, lactoperoxidase is the next most abundant of the cationic proteins captured by ion exchange from milk (0.03-0.075 mg/ml milk). In a salt gradient lactoperoxidase elutes from cation exchange before lactoferrin at 25-30 mS.

The growth factors EGF, IGF 1, IGF 2, TGF B1 and TGF B2 are present in milk in ng/ml quantities, and have been shown to be captured by cation exchange.

A number of other biologically active cationic peptides elute between lactoperoxidase and lactoferrin at 35-40 mS (intermediate fraction). These are likely to include quiescin, jacalin-like protein, and lysozyme-like proteins, such as chitinase-like protein (CLP-1) or lysosomal alpha mannosidase (LAM). Therefore the concentration of salt used at each step in the elution determines whether these biologically active peptides are in the lactoperoxidase fraction or the lactoferrin fraction. In preliminary studies, the inventors have identified that the whole cationic fraction appears to have a much higher level of selectivity compared to just proteins in the intermediate fraction.

Immunoglobulins are eluted in low salt (15-20 mS).

In a preferred embodiment the milk, or milk product may be passed through a membrane having cationic exchange properties, or a column packed with the cationic exchange resin or a batch reactor with suspended cationic resin, whereby the micro-components adsorb from the starting milk or product thereof onto the cationic exchange resin or membrane.

After adsorption of milk micro-components the cationic fraction is preferably extracted by elution with a salt solution.

However, this should not be seen as limiting as elution of the cationic fraction may also be via a shift in pH. This method, however, is not popular in large scale commercial processes as the high pH required to remove lactoferrin from the resin could be damaging to the lactoferrin, or in the present case any other components in the cationic fraction.

In a preferred embodiment, before elution, the resin or membrane may be rinsed with a salt solution. Preferably the rinse solution may be sodium chloride or sodium bicarbonate, with conductivity between 5 and 10 mS (millisiemens/cm). This rinse step ensures that substantially all non-adsorbed milk components are rinsed off the resin or out of the membrane.

In a preferred embodiment the cationic fraction may be eluted in a salt gradient between substantially 10 mS and 100 mS conductivity (0.1 to 2.0 M salt).

In a preferred embodiment the cationic fraction may be eluted in a single fraction by passing a salt solution with conductivity between 80 and 100 mS through the column or membrane.

In a preferred embodiment the elution salt may preferably be sodium chloride. However, this should not be seen as limiting as other salts including sodium acetate, sodium bicarbonate, ammonium bicarbonate, or potassium chloride may be used.

Having the cationic fraction eluted in a one-step elution provides a significant advantage. It decreases the length of extraction time thereby decreasing the possibility of bioactives being denatured. It also decreases the time, labour and cost of the extraction process. This can provide a significant advantage, especially on a large scale. Furthermore, the results clearly show that inhibitory effect (and we also expect selectivity) will be enhanced when the components of milk having a pl above 6.8 are retained as a single isolated fraction and administered together.

In a preferred embodiment after initial monitoring of the protein levels in the eluted stream to determine the concentration of salt and the volumes required to elute all the protein, the typical large scale process operates on volumes rather than continuous monitoring.

In a preferred embodiment the extraction may be undertaken in a continuous manner.

In another preferred embodiment, the extraction may be undertaken in a batch elution.

In the above preferred embodiments the cationic fraction may be extracted by a ‘one-step’ process, by step elution.

In an alternative embodiment the cationic fraction may be extracted using a gradient elution.

However this should not be seen as limiting as the cationic fraction may also be extracted in independent fractions and recombined to form the complete cationic fraction at a later stage.

In some embodiments the cationic fraction may undergo further treatments, by standard techniques known in the art, for example, to remove salt, or to concentrate, or to filter for sterility or to remove endotoxin. The concentrated fraction may also be lyophilised.

In a preferred embodiment the cationic fraction may be concentrated to approximately 20% solids.

In the case of the cationic fraction being extracted from milk that is processed in the usual manner involving storage, transport and conversion to skim milk or whey the temperature should preferably be maintained at substantially 4-7° C. to minimize microbial growth.

In the case of the cationic fraction being extracted from whole milk the temperature should preferably be maintained at not less than 35° C. to ensure that lipids remain in a liquid state so that they can easily pass through the extraction material. And to ensure the bioactivity of the factors in the cationic fraction are maintained at or close to the endogenous state.

In an alternative embodiment the cationic fraction may be extracted from genetically modified animals, for example genetically modified enhancement of lactoferrin production in dairy cows. One skilled in the art would realise that extraction from the milk of genetically modified animals may affect the ratio or concentrations of lactoferrin, or other components in the cationic fraction, or a whole cascade of key components.

In one preferred embodiment the cationic fraction may be extracted from the same species of animal that the treatment substance is intended to be used on.

SUMMARY OF ADVANTAGES

The present invention is believed to provide a number of significant advantages including:

• • High selectivity towards inhibition of a wide number of pathogenic microorganisms without a similar level of inhibition towards a wide number of commensals;
  • Potential for ability of combination, such as the composition, to target opportunistic commensals;
  • Likelihood of further synergies with other components such as pre-biotics or probiotics;
  • Low concentration of lactoperoxidase in the combination needed to achieve desired effect, so as to avoid likelihood of issues such as hemolysis.
  • The synergy observed towards improved selectivity appears to be closely linked to the retention of lactoperoxidase together with other protein(s) in the cationic fraction of milk with a pl above 6.8, which also conveniently is beneficial in terms of ease of manufacture, improved stability, and consumer acceptance (low processing required). This is contrary to U.S. Pat. No. 5,888,505 which highlighted purified forms of peroxide (e.g. myeloperoxidase) together with added halides/co-factors are needed to achieve a desired result, and further that lactoperoxidase essentially had no ability to provide selectivity between pathogens and commensals.
  • The ability to provide a cationic fraction eluted in a one-step elution decreases the length of extraction time required for extraction, thereby decreasing the possibility of bioactives being denatured. It also decreases the time, labour and cost of the extraction process. This can provide a significant advantage, especially on a large scale. It also appears to improve inhibitory effects, and we expect retention of the selectivity profiles.
  • The combination, such as the composition, may be prepared conveniently from milk and is therefore considered to be natural and safe to use.
  • Unlike broad spectrum antibiotics (which will kill all the microorganisms in the microbiome), the cationic fraction naturally preserves the beneficial commensals.
  • Unlike probiotics or prebiotics, the combination is potent at inhibiting pathogenic microorganisms and therefore will have a better therapeutic effect for treating existing infections (not just being a preventative).
  • Equally, the potency of the combination does not rule out the ability to use the combination in a preventative manner to boost, modulate or maintain a person's (or other animal's) microbiome to avoid infections, conditions or diseases from transpiring in future.

EXAMPLES

Further aspects of the present invention will become apparent from the following description which is given by way of example only.

Example 1: Assessment of the Proteins in the Composition (i.e. The Cationic Fraction) Via Mass Spectrometry

The process of producing the cationic fraction involved fractionating milk through a cation exchange resin, eluting the bound components from the resin using a salt solution, which can be either a one-step high molarity (>1M) salt or a gradient elution from a lower molarity up to over 1M, collecting the eluted components in a single fraction, and then desalting and purifying the collected fraction.

The cationic fraction was analysed for its constituent components, and the results shown in Table 2. This shows a typical result for yield and identity of the major proteins identified in the cationic protein fraction.

This particular cationic fraction was captured from raw, whole milk.

TABLE 2
Major sub-fractions from the cationic fraction, as measured by Mass
Spectrometry (MS). (Lactoperoxidase was determined via extinction
coefficient rather than MS.) Without wishing to be bound by theory
it is believed that the selective antimicrobial activity detailed
herein may be due to the inclusion of lactoperoxidase, together
with at least one other component in the combination. The selective
antimicrobial activity may be enhanced by the presence of at least
one or more of lactoferrin, angiogenin, and/or lysozyme-like protein.
The selective antimicrobial activity may be enhanced by the presence
of at least one or more of lysozyme-like protein, quiescin, and
jacalin-like protein. The selective antimicrobial activity may be
enhanced by the presence of at least one or more minor sub-fractions,
such as cathlecidin-1 and/or serum amyloid A.

	Total Protein	% of total of major	Isoelectric
Identity from MS	(mg/ml)	sub-fractions	point

lactoperoxidase	4.2	8.0%	8.3
quiescin	1.6	3.0%	8.69
jacalin-like protein	1.4	2.7%	8.71
chitinase-like protein	0.4	0.8%	8.74
angiogenin	10.0	19.0%	9
Lactoferrin	35.0	66.5%	8.7
TOTAL	52.6	100.0%

Example 2: Inhibition Trials on Pathogens Vs Commensals

The Applicant used the methodology described herein to prepare a cationic fraction isolated from bovine milk as described in Table 2. The composition was tested in vitro against a number of microorganisms using micro-titre plates and some in agar diffusion tests. The results shown in Table 3 (shown below) identify the MIC (mg/ml) of the cationic fraction against the different microorganisms using an aerobic, microtiter plate based method.

TABLE 3
Inibitory analysis of cationic fraction against
range of commensals and pathogens.

	MIC
	mg/ml

	Pathogens
	Propionibacterium acnes	0.1
	Trichophyton mentagrophytes	0.1
	Trichophyton rubrum	0.1
	Escherichia coli	0.1
	Streptococcus pyogenes	0.1
	Malassezia furfur	0.2
	Commensal/Opportunistic pathogen
	Candida albicans	0.5
	Streptococcus mutans	2.5
	Staphylococcus aureus	3
	Commensals and Probiotics
	Streptococcus salivarius (probiotic strain)	>5
	Streptococcus pneumonia	>10
	Staphylococcus epidermidis	>10
	Staphylococcus hominis	>10
	Lactobacillus bulgaricus	>10
	Lactobacillus casei	>10
	Porphyromonas gingivalis*	>10
	*In humans in particular, Porphyromonas gingivalis is regarded as a commensal organism. However, Porphyromonas gingivalis has been implicated in periodontal disease in canines (esp. dogs) and could be regarded as a pathogen in that Family.

The conclusions that can be reached from this preliminary work are:

• • 1. Microorganisms that frequently cause infections (i.e. pathogens) are killed by the lowest concentrations of the combination (provided as a composition).
  • 2. Microorganisms that are common, harmless commensals and/or are used as probiotics were not killed by the highest concentrations tested in these trials. (100× greater than the concentration that killed pathogens).
  • 3. Intermediate organisms, such as Candida albicans, which are frequently found as harmless commensals and only cause infection (i.e. become opportunistic) when the conditions change in the local environment so that growth is enhanced (e.g an increase in sugar concentration) show moderate to high MIC values.

Example 3: Comparative Selectivity Between Isolated and/or Recombined Proteins, Sub-Cationic Fractions, and Whole Cationic Fraction

Additional studies were conducted which show the individual proteins in the cationic fraction (i.e. lactoperoxidase, lactoferrin, quiescin-like, jacilin-like, chitisase-like, angiogenin) have poor anti-microbial effectiveness against pathogens, and therefore will not be able to provide the selectivity offered by the combination of proteins in the preferred compositions (most preferably the full suite of proteins in the cationic fraction). The results are shown in FIGS. 5 and 6.

Informal results also showed that the middle cationic fraction (not containing lactoperoxidase or lactoferrin) has relatively poor selectivity, supporting that lactoperoxidase is an important component of the composition, but requires other protein(s) from milk in order to develop the selectivity profile observed.

Also, the results shown in FIGS. 5 and 6 showed that with there is a benefit of retaining the proteins together during the extraction process, rather than isolating them and recombining to form the composition. Nonetheless, the recombined fraction provides useful, albeit not optimal, inhibition of E. coli and S. aureus.

The fact that anti-microbial activity is enhanced when the cationic fraction remains intact also strongly suggests the selectivity will be better if the components are not individually separated from one another before recombining. That is, the combination is preferably provided as a composition which preferably includes the cationic fraction of milk.

Based on these results, the following schematic representation is provided to illustrate the effectiveness of the present invention towards selectivity.

	MIC (mg/ml)

Low

High

	<0.1	0.5	1.0	2.0	5.0	>10.0

Lactoferrin	Pathogenic-Opportunistic-
(Lf)	Commensals (no selectivity)

Lactoperoxidase			Pathogenic-Opportunistic-
(Lp)			Commensals (no selectivity)
Middle Cationic			Pathogenic-Opportunistic-
Fraction*			Commensals (no selectivity)

Whole cationic

Pathogenic

Opportunistic

Commensals

fraction**		commensals such
		as C albicans
*e.g. angiogenin, quiescin, jacalin-like protein, and chitinase-like protein - no Lp or Lf
**as seen in Table 4.

Example 4: Effect of Additional Substrates

FIGS. 3 and 4 illustrate that anti-microbial effects (and hence potentially selectivity too) are improved in the presence of substrates. If such substrates are not present at the site of infection, it may be beneficial to include suitable substrates within the combination of the invention. The extent of growth of the microorganism is indicated by the height of the bars. The shortest bars show maximum inhibition of growth. For this figure, the left-hand bars indicate that some inhibition of growth is achieved with the cationic fraction alone at a concentration of 1 mg/ml. However, adding 40 ppm of sodium thiocyanate to the cationic fraction allowed total growth inhibition to occur at a cationic fraction concentration of 2 mg/ml. This indicates that lactoperoxidase contributes to the antimicrobial activity when its substrate (thiocyanate) is included.

FIG. 4 shows the results of a different formulation of the cationic fraction against Streptococcus uberis, this time using sodium thiocyanate (75 ppm) and ascorbate (150 ppm) as substrates. Against Streptococcus uberis, there is no inhibition in vitro using the cationic fraction alone up to 0.8 mg/ml. However, adding sodium thiocyanate and ascorbate shows an inhibitory effect occurring as low as 0.2 mg/ml of the cationic fraction. This confirms that in the absence of milk (or another natural source of substrates) the addition of thiocyanate (as substrate) and ascorbate (as a source of peroxide) may be useful for increasing inhibition (and perhaps selectivity) towards Streptococcus uberis.

Note that in FIG. 4, none of the additives were totally inhibitory on their own. The samples labeled ‘0’ in the figure are buffer-only and additive-only samples.

TABLE 4
Example formulations used for comparative
testing are provided below

Combination				“Activated
(as a	Lactoper-	Lacto-	“Cationic	cationic
composition)	oxidase	ferrin	fraction”	fraction”

Lactoferrin	0%	92.9%	64.3%	61.3%
Lactoperoxidase	97.7%*	0%	22.8%	26.6%
Other protein	<2.3%* (n.m.)	5.1%	9.3%	8.1%
Glucose	0%	0%	0%	0.845%
Glucose oxidase	0%	0%	0%	0.015%
Thiocyanate	0.004%	0%	0.004%	0.004%
Monolaurin	0%	0%	0%	0.25%
*total protein value as no specific assay for lactoperoxidase is reported for this material, hence other protein level cannot be estimated.
Note:
for each sample, the remainder of material to 100% is largely inorganic (measured as ash) or residual moisture.
Note:
n.m. was not measured
Note:
there are hundreds or probably thousands of minor proteins (“other protein”) within the cationic whey fraction.
Note:
the “activated cationic fraction” includes glucose, glucose oxidase and monolaurin which are not typically found in milk, let alone the “cationic fraction”. Glucose oxidase can use glucose as a substrate to generate peroxide in situ. Other peroxide generating systems may include percarbonate or peracetate, which may be encapsulated or coated to control the release rates of the peroxides. These components may be considered to act as adjuvants.

Thiocyanate is present in the “activated cationic fraction” and is an example of a substrate. Other examples of substrates include iodide or chloride, having countercations of sodium, potassium or calcium.

The innate lactoperoxidase system protects the eyes, nose, mouth and airways from invasion by harmful microbes and requires presence of the lactoperoxidase enzyme, peroxide and thiocyanate or halide.

H₂O₂ is naturally present in internal biological environments as it is a by-product of various oxidative processes. For example, neutrophils produce large amounts of free peroxy radicals (O₂⁻) of which the steady state concentration has been estimated to be in the micromolar range. (Ref. Hampton, M B, Kettle A J, Winterbourn C C. Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing. Blood 1998; 92:3007-17)

Peroxidases (such as lactoperoxidase) are present in biological secretions and catalyse H₂O₂ dependent oxidation of halides (thiocyanate, iodide, bromide, chloride) that can react with and kill microbes. (Ref. Klebanoff S J. Antimicrobial mechanisms in neutrophilic polymorphonuclear leukocytes. Semin. Hematol 1975; 12:117-42)

Thiocyanate is naturally present in lymph and blood, in the mammary, salivary and thyroid glands and their secretions, in synovial, cerebral, cervical and spinal fluids and in organs such as stomach and kidney. For example, thiocyanate levels measured in human trachea-bronchial secretions from intubated adult patients were 0.46+/−0.19 mM or 26.7+/−11 ppm (range 16-38 ppm). (Ref. Wijkstrom-Frei, C., El-Chemaly, S., Ali-Rachedi, R., Gerson, C., Cobas, M. A., Forteza, R., Salathe, M. and G. E. Conner. 2003. Lactoperoxidase and human airway host defense, Am. J. Respir. Cell Mol. Biol., 29:206-12).

As such, in some circumstances, such as where the combination is being applied internally, to an open wound, or to a mucosal membrane it may not be necessary, or even preferable to provide an adjuvant and/or a substrate as part of the combination since that substrate will be provided endogenously by the tissue to which the combination is applied.

In other circumstances the endogenous concentration of the adjuvant and/or substrate may be too low or non-existent to have an appreciable effect on the activity of the combination. In those circumstances it may be preferable to include an adjuvant and/or substrate in the combination.

For in vitro testing, the assay medium will not typically contain the adjuvant and/or substrate and the improved results for the “activated cationic fraction” compared with the “cationic fraction” may be partially explained by the beneficial effect of the substrate and adjuvants contained in the activated cationic fraction. However, the “cationic fraction” may still provide useful selectivity when applied, for example, to an area of the body where the substrate and/or adjuvants (halides and peroxide generation, for example) are already present such as application internally, to an open wound, or to a mucosal membrane.

Example 5: Bacterial Selectivity

The activity and selectivity of a range of test compounds/compositions were determined against a range of pathogenic and commensal organisms.

The methodology for each of the following test compositions is described below:

• • Lactoperoxidase (sample 3);
  • Lactoferrin (sample 2);
  • “Cationic fraction” (sample 4); and
  • “Activated cationic fraction” (sample 1)

Each sample was prepared as a stock solution at 5 mg/ml. Samples 1, 3 and 4 were dissolved in HBSS which contains potassium isothiocyanate at 40 ppm (40 μg/ml). Sample 2 was dissolved in HBSS at 5 mg/ml (Table 3).

Aerobic Testing

Experiment Protocol for C. albicans, S. aureus, S. epidermidis, S. mitis, S. mutans and S. salivarius

• • 1. For C. albicans Sabouraud dextrose broth powder was added to distilled water at 30 g/L and stirred. For S. aureus and S. epidermidis tryptic soy broth powder was added to distilled water at 30 g/L and stirred. For S. mitis, S. mutans and S. salivarius 5% sheep blood broth was prepared by diluting the sheep blood with double distilled water.
  • 2. The broth solutions were then boiled for 1 minute with stirring to completely dissolve the powder.
  • 3. The broth media were then autoclaved at 121° C. for 20 minutes.
  • 4. A small quantity of each pure microorganism was taken and used to inoculate 40 ml of broth medium. The inoculated broth was incubated for approximately 66 hours at 37° C.
  • 5. The broth culture was diluted with fresh, sterile broth medium to an OD_650nm of approximately 0.1, equivalent to approx 10⁵ CFU/ml prior to commencement of MIC testing. This is the inoculant which will be used to inoculate the test wells in each plate. The inoculant was held at 4° C. until required for plating.
  • 6. Stock solutions of the test sample were prepared such that the concentration is 5 mg/ml in the appropriate broth.
  • 7. The reference antibiotic was dissolved in broth to give a final concentration of 100 μg/ml.
  • 8. 96 well microtitre plates were then set up as indicated in the plate layout diagrams below: 200 μl of the appropriate sample stock solution of test sample in the appropriate broth, antibiotic standards and vehicle control were added to the relevant wells in Column 1 on Plates 1 to 3.
  • 9. To all other wells 100 μl of the appropriate sterile broth was added.
  • 10. Using a multichannel pipette, 100 μl of the sample and antibiotic was sampled from the wells of column 1 on each plate and transferred to wells in column 2, mixed thoroughly by pipetting up and down 5 times. Fresh tips were added to the pipette and 100 μl of solution was transferred from the wells of column 2 to those of column 3, mixed thoroughly by pipetting up and down 5 times and then discarding the tips. This process was continued through to the wells of column 11 on each plate. This process will result in serial double-dilutions that range from 5 mg/ml to 0.005 mg/ml for samples, and 100 μg/ml to 0.098 μg/ml for the antibiotic standard. The 12th and final well in each row (Plates 1 to 3) contain wells of broth only with inoculants and broth only without inoculants (Plates 4). These wells serve as sterility control blanks and test substance free control blanks respectively.
  • 11. Wells A1-12, B1-12, C1-12, D1-12, E1-12, F1-12, G1-12 and H1-12 on Plate 4 contain broth only and were not inoculated with seed culture. These wells served as sterility controls and blank for each row. Wells A12-F12 (Plates 1-2) and A12-C12 (Plate 3) contained the cells and serve as the negative control.
  • 12. 100 μl of inoculant or broth were added to each well as indicated in the plate layouts below. The addition of inoculant or broth halve the extract concentration in each well giving final well concentrations ranging from 2.5 mg/ml to 0.0025 mg/ml for samples (including vehicle control) and 50 μg/ml to 0.049 μg/ml for the antibiotic standard.
  • 13. The plates were gently tapped to ensure even mixing of the inoculant with the sample solutions.
  • 14. The OD_650nm of each well were read using a Versamax microtitre plate reader. This will be recorded as the zero time reading.
  • 15. The plates were incubated for 3 hours at 37° C. at which time the OD_650nm of each well were read and recorded as the 3 hour reading.
  • 16. The plates were returned to the incubator for a further 13 hours and the OD_650nm of each well was read and recorded as the 16 hour reading.
  • 17. The microtitre plates were returned to the incubator for a further 8 hours and the OD_650nm was read and recorded as the 24 hour reading.
  • 18. Once the OD_650nm of the plates was read, the wells containing the highest dilution of each sample (lowest concentration of test extract) without a detectable change in OD_650nm in comparison to the initial reading at time zero were noted.

Anaerobic Testing

Experiment Protocol for B. bifidum, B. breve, C. difficile, C. perfringens and P. acnes

• • 1. For each of these bacteria, Brain Heart Infusion Blood broth was used. It was prepared by adding it to distilled water at 37 g/L.
  • 2. The broth solution was then boiled for one minute with stirring to completely dissolve the powder.
  • 3. The broth media was then autoclaved at 121° C. for 20 minutes.
  • 4. A small quantity of each organism was used to inoculate 40 ml of the Brain Heart Infusion broth that was de-aerated by bubbling nitrogen into it. This sealed tube is then incubated at 37° C.
  • 5. During this incubation, the samples were prepared. Stock solutions of the samples were prepared at 5 mg/ml in the broth.
  • 6. The reference antibiotic was dissolved in broth to give a final concentration of 100 μg/ml.
  • 7. 96 well microtitre plates was then set up as indicated in the plate layout diagrams below: 200 μl of the appropriate sample stock solution of test sample in the appropriate broth, antibiotic standards and vehicle control) was added to the relevant wells in Column 1 on Plates 1 to 3.
  • 8. To all other wells 100 μl of the appropriate sterile broth was added.
  • 9. Using a multichannel pipette, 100 μl of the sample and antibiotic was sampled from the wells of column 1 on each plate and transferred to wells in column 2, mixed thoroughly by pipetting up and down 5 times. Fresh tips were added to the pipette and 100 μl of solution was transferred from the wells of column 2 to those of column 3, mixed thoroughly by pipetting up and down 5 times and then discarding the tips. This process was continued through to the wells of column 11 on each plate. This process results in serial double-dilutions that range from 5 mg/ml to 0.005 mg/ml for samples, and 100 μg/ml to 0.098 μg/ml for the antibiotic standard. The 12th and final well in each row (Plates 1 to 3) contain wells of broth only with inoculants and broth only without inoculants (Plates 4). These wells serve as sterility control blanks and test substance free control blanks respectively.
  • 10. Wells A1-12, B1-12, C1-12, D1-12, E1-12, F1-12, G1-12 and H1-12 on Plate 4 contain broth only and are not inoculated with seed culture. These wells serve as sterility controls and blank for each row. Wells A12-F12 (Plates 1-2) and wells A12-C12 (Plate 3) contain the cells and serve as the negative control.
  • 11. 100 μl of inoculant or broth are added to each well as indicated in the plate layouts below. The addition of inoculant or broth halve the extract concentration in each well giving final well concentrations ranging from 2.5 mg/ml to 0.0025 mg/ml for samples (including vehicle control) and 50 μg/ml to 0.049 μg/ml for the antibiotic standard.
  • 12. The plates are gently tapped to ensure even mixing of the inoculant with the sample solutions.
  • 13. The OD_650nm of each well were read using a Versamax microtitre plate reader. This was recorded as the zero time reading.
  • 14. The plates are then placed in a sealed container along with one or more anaerobic pouches. The sealed container is then incubated at 37° C.
  • 15. After 3 hours, the plates are removed from the container and the OD_650nm of each well are read in the platereader immediately, one plate at a time. The plates were then returned to the container along with new anaerobic pouches and plates incubated at 37° C.
  • 16. At 16 hours after commencing the study, step 15 was repeated.
  • 17. After 24 hours, the OD_650nm of each well was measured.
  • 18. Once the OD_650nm of the plates was read, the wells containing the highest dilution of each sample (lowest concentration of test extract) without a detectable change in OD_650nm in comparison to the initial reading at time zero was noted.

The results of the testing of the anaerobic species and the aerobic species are presented in Table 5.

TABLE 5
MIC values @ 3 hrs (mg/ml)

Organism

	Candida	Staphylococcus	Staphylococcus
	albicans	aureus	epidermidis
Type	Commensal/	Pathogen	Commensal
	Opportunistic
	Pathogen
Sample 1 Activated	0.005	0.002	0.01
cationic fraction
Selectivity*	2.0	5.0	1.0
Sample 2 Lactoferrin	0.625	≥2.5	≥2.5
Selectivity*	≥4.0	—	1.0
Sample 3 Lactoperoxidase	≥2.5	≥2.5	≥2.5
Selectivity*	—	—	1.0
Sample 4 Cationic fraction	≥2.5	≥2.5	≥2.5
Selectivity*	—	—	1.0

MIC values @ 48 hrs (mg/ml)

Organism

	Streptococcus	Streptococcus	Streptococcus
	mitis	mutans	salivarius
Type	Commensal	Pathogen	Commensal
Sample 1 Activated	0.625	0.002	0.156
cationic fraction
Selectivity*	1.0	78.0-300**	1.0
Sample 2 Lactoferrin	1.25	≥2.5	≥2.5
Selectivity*	≥2.0	—	1.0
Sample 3 Lactoperoxidase	≥2.5	≥2.5	≥2.5
Selectivity*	—	—	1.0
Sample 4 Cationic fraction	≥2.5	≥2.5	≥2.5
Selectivity*	—	—	1.0
*1.0 = no selectivity
**relative to S. salivarius and S. mitis

Example 6: Further Bacterial Selectivity—Canine Microbiome

The Activity and Selectivity of the Combination of the Invention Against 4 Bacterial Strains Isolated from Dog Saliva

Single bacterial colonies were grown on Columbia blood agar (CBA). A cotton tipped swab was dipped in fresh dog saliva and swabbed to CBA plates. The plates were incubated anaerobically for 24 hours at 37° C. Four single colonies were then streaked to fresh CBA and incubated anaerobically for a further 24 hours. Two haemolytic colonies and two non-haemolytic colonies were selected. The haemolytic colonies are more likely to be pathogenic and the non-haemolytic colonies are more likely to be commensals.

Lawns of the four isolates were created using cotton tipped swabs on CBA plates. A suspension of the combination of the invention (50 mg/ml) was prepared. Three halving dilutions were made from the suspension, and 25 ul drops of the four dilutions were added to the plates. The plates were incubated anaerobically for 18 hours. The size of any zones of clearing were measured using calipers.

The combination of the invention only inhibited the haemolytic bacterial strains as shown in Table 6.

TABLE 6
Inhibition of bacteria isolated from canine saliva. Isolates
1 and 2 are haemolytic, isolates 3 and 4 are non-haemolytic.
N = no clearing, P = only some colonies grew.

Diameter clearing (mm)

	50	25	12.5	6.25
	mg/mL	mg/mL	mg/mL	mg/mL

Isolate 1 (haemolytic)	9.1	6.9	6.8	N
Isolate 2 (haemolytic)	8.3	6.7	5.7	N
Isolate 3 (non-haemolytic)	N	N	N	N
Isolate 4 (non-haemolytic)	N	N	N	N

The procedure was repeated using human saliva. Two haemolytic strains (1 and 2) were inhibited, and two non-haemolytic strains (3 and 4) were able to grow in the presence of the combination of the invention, at the concentrations tested, as shown in Table 7. A slightly higher concentration of the combination of the invention was required to inhibit the human derived bacteria than the canine derived bacteria.

TABLE 7
Inhibition of bacteria isolated from human saliva. Isolates
1 and 2 are haemolytic, isolates 3 and 4 are non-haemolytic.
N = no clearing, P = only some colonies grew.

Diameter clearing (mm)

	50	25	12.5	6.25
	mg/mL	mg/mL	mg/ml	mg/mL

Isolate 1 (haemolytic)	14.2	13.2 (P)	N	N
Isolate 2 (haemolytic)	13.7	N	N	N
Isolate 3 (non-haemolytic)	N	N	N	N
Isolate 4 (non-haemolytic)	N	N	N	N

Example 7: Further Bacterial Selectivity—Canine Microbiome (A. actinomycetemcomitans, L. acidophilus, S. mutans, and P. gingivalis Incubated Anaerobically)

Using a similar growth methodology to that described in Example 6, A. actinomycetemcomitans (incubated anaerobically) was exposed to the combination of the invention at the same dilutions. Four drops of the combination of the invention were placed on the surface of each plate. The plates are shown in FIG. 7. The treated regions are as follows:

• • Lower right 50 mg/mL dilution
  • Lower left 25 mg/ml dilution
  • Upper left 12.5 mg/ml dilution
  • Upper right 6.25 mg/ml dilution

The results indicate that the growth of A. actinomycetemcomitans was potentially affected by each of the dilutions, in a concentration dependent manner, although the effected was most pronounced for the two more concentrated dilutions.

The same methodology was applied to each of L. acidophilus, S. mutans, and P. gingivalis, the results quantified (size of clearings on the plate) and the combined results shown below in Table 8. The results shown were obtained using an anaerobic agar plate method, which is not directly comparable to the aerobic, microtiter plate based method used to produce the data in Table 3:

TABLE 8
Inhibition of anaerobic bacteria. The number represent
the size of clearings on agar plates, such that the
bigger the clearing the higher the antimicrobial activity.
P = partial clearing, Ob = obscured by conjugate.

Diameter clearing (mm)

	50	25	12.5	6.25
	mg/ml	mg/mL	mg/mL	mg/ml

L. acidophilus	0	0	0	0
S. mutans	11.7	10.8	0	0
A. actinomycetemcomitans	13.7	10.6	0	0
P. gingivalis	11.9	11.4	10.8	P

P. gingivalis and A. actinomycetemcomitans have been associated with periodontal disease in dogs, and in the canine oral microbiome may be regarded as pathogenic.

L. acidophilus is given to humans and dogs as a probiotic, primarily to improve gut health. It is a commensal/beneficial bacteria.

As the results show, selective inhibition of pathogenic bacteria in the canine oral microbiome can be advantageously realised by administration of the combination of the invention.

Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope thereof.

All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art, in New Zealand or in any other country.

It is acknowledged that the term ‘comprise’ may, under varying jurisdictions, be attributed with either an exclusive or an inclusive meaning. For the purpose of this specification, and unless otherwise noted, the term ‘comprise’ shall have an inclusive meaning—i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non-specified components or elements. This rationale will also be used when the term ‘comprised’ or ‘comprising’ is used in relation to one or more steps in a method or process.